Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis

This work is licensed under a Creative Commons Attribution 4.0 International License.The use of extracellular vesicles, specifically exosomes, as carriers of biomarkers in extracellular spaces has been well demonstrated. Despite their promising potential, the use of exosomes in the clinical setting is restricted due to the lack of standardization in exosome isolation and analysis methods. The purpose of this review is to not only introduce the different types of extracellular vesicles but also to summarize their differences and similarities, and discuss different methods of exosome isolation and analysis currently used. A thorough understanding of the isolation and analysis methods currently being used could lead to some standardization in the field of exosomal research, allowing the use of exosomes in the clinical setting to become a reality

Quantification of Flavin-containing Monooxygenases 1, 3, and 5 in Human Liver Microsomes by UPLC-MRM-Based Targeted Quantitative Proteomics and Its Application to the Study of Ontogeny

Flavin-containing monooxygenases (FMOs) have a significant role in the metabolism of small molecule pharmaceuticals. Among the five human FMOs, FMO1, FMO3, and FMO5 are the most relevant to hepatic drug metabolism. Although age-dependent hepatic protein expression, based on immunoquantification, has been reported previously for FMO1 and FMO3, there is very little information on hepatic FMO5 protein expression. To overcome the limitations of immunoquantification, an ultra-performance liquid chromatography (UPLC)-multiple reaction monitoring (MRM)-based targeted quantitative proteomic method was developed and optimized for the quantification of FMO1, FMO3, and FMO5 in human liver microsomes (HLM). A post-in silico product ion screening process was incorporated to verify LC-MRM detection of potential signature peptides before their synthesis. The developed method was validated by correlating marker substrate activity and protein expression in a panel of adult individual donor HLM (age 39–67 years). The mean (range) protein expression of FMO3 and FMO5 was 46 (26–65) pmol/mg HLM protein and 27 (11.5–49) pmol/mg HLM protein, respectively. To demonstrate quantification of FMO1, a panel of fetal individual donor HLM (gestational age 14–20 weeks) was analyzed. The mean (range) FMO1 protein expression was 7.0 (4.9–9.7) pmol/mg HLM protein. Furthermore, the ontogenetic protein expression of FMO5 was evaluated in fetal, pediatric, and adult HLM. The quantification of FMO proteins also was compared using two different calibration standards, recombinant proteins versus synthetic signature peptides, to assess the ratio between holoprotein versus total protein. In conclusion, a UPLC-MRM-based targeted quantitative proteomic method has been developed for the quantification of FMO enzymes in HLM

Antiarrhythmic and proarrhythmic effects of subcutaneous nerve stimulation in ambulatory dogs

Background High output subcutaneous nerve stimulation (ScNS) remodels the stellate ganglia and suppresses cardiac arrhythmia. Objective To test the hypothesis that long duration low output ScNS causes cardiac nerve sprouting, increases plasma norepinephrine concentration and the durations of paroxysmal atrial tachycardia (PAT) in ambulatory dogs. Methods We prospectively randomized 22 dogs (11 males and 11 females) into 5 different output groups for 2 months of ScNS: 0 mA (sham) (N=6), 0.25 mA (N=4), 1.5 mA (N=4), 2.5 mA (N=4) and 3.5 mA (N=4). Results As compared with baseline, the changes of the durations of PAT episodes per 48 hours were significantly different among different groups (sham, -5.0±9.5 s; 0.25 mA 95.5±71.0 s; 1.5 mA, -99.3±39.6 s; 2.5 mA, -155.3±87.8 s and 3.5 mA, -76.3±44.8 s, p<0.001). The 3.5 mA group had greater reduction of sinus heart rate than the sham group (-29.8±15.0 bpm vs -14.5±3.0 bpm, p=0.038). Immunohistochemical studies showed that the 0.25 mA group had a significantly increased while 2.5 mA and 3.5 mA stimulation had a significantly reduced growth-associated protein 43 nerve densities in both atria and ventricles. The plasma Norepinephrine concentrations in 0.25 mA group was 5063.0±4366.0 pg/ml, which was significantly higher than other groups of dogs (739.3±946.3, p=0.009). There were no significant differences in the effects of simulation between males and females. Conclusions In ambulatory dogs, low output ScNS causes cardiac nerve sprouting, increases plasma norepinephrine concentration and the duration of PAT episodes while high output ScNS is antiarrhythmic

Cellular Target Deconvolution of Small Molecules Using a Selection-Based Genetic Screening Platform

Small-molecule drug target identification is an essential and often rate-limiting step in phenotypic drug discovery and remains a major challenge. Here, we report a novel platform for target identification of activators of signaling pathways by leveraging the power of a clustered regularly interspaced short palindromic repeats (CRISPR) knockout library. This platform links the expression of a suicide gene to the small-molecule-activated signaling pathway to create a selection system. With this system, loss-of-function screening using a CRISPR single-guide (sg) RNA library positively enriches cells in which the target has been knocked out. The identities of the drug targets and other essential genes required for the activity of small molecules of interest are then uncovered by sequencing. We tested this platform on BDW568, a newly discovered type-I interferon signaling activator, and identified stimulator of interferon genes (STING) as its target and carboxylesterase 1 (CES1) to be a key metabolizing enzyme required to activate BDW568 for target engagement. The platform we present here can be a general method applicable for target identification for a wide range of small molecules that activate different signaling pathways

Parallel Bayesian probabilistic integration for structural reliability analysis with small failure probabilities

Bayesian active learning methods have emerged for structural reliability analysis and shown more attractive features than existing active learning methods. However, it remains a challenge to actively learn the failure probability by fully exploiting its posterior statistics. In this study, a novel Bayesian active learning method termed ‘Parallel Bayesian Probabilistic Integration’ (PBPI) is proposed for structural reliability analysis, especially when involving small failure probabilities. A pseudo posterior variance of the failure probability is first heuristically proposed for providing a pragmatic uncertainty measure over the failure probability. The variance amplified importance sampling is modified in a sequential manner to allow the estimations of posterior mean and pseudo posterior variance with a large sample population. A learning function derived from the pseudo posterior variance and a stopping criterion associated with the pseudo posterior coefficient of variance of the failure probability are then presented to enable active learning. In addition, a new adaptive multi-point selection method is developed to identify multiple sample points at each iteration without the need to predefine the number, thereby allowing parallel computing. The effectiveness of the proposed PBPI method is verified by investigating four numerical examples, including a turbine blade structural model and a transmission tower structure. Results indicate that the proposed method is capable of estimating small failure probabilities with superior accuracy and efficiency over several other existing active learning reliability methods

Adapting Multi-Lingual ASR Models for Handling Multiple Talkers

State-of-the-art large-scale universal speech models (USMs) show a decent automatic speech recognition (ASR) performance across multiple domains and languages. However, it remains a challenge for these models to recognize overlapped speech, which is often seen in meeting conversations. We propose an approach to adapt USMs for multi-talker ASR. We first develop an enhanced version of serialized output training to jointly perform multi-talker ASR and utterance timestamp prediction. That is, we predict the ASR hypotheses for all speakers, count the speakers, and estimate the utterance timestamps at the same time. We further introduce a lightweight adapter module to maintain the multilingual property of the USMs even when we perform the adaptation with only a single language. Experimental results obtained using the AMI and AliMeeting corpora show that our proposed approach effectively transfers the USMs to a strong multilingual multi-talker ASR model with timestamp prediction capability.Comment: Accepted by Interspeech 202

Sex‐specific activation of SK current by isoproterenol facilitates action potential triangulation and arrhythmogenesis in rabbit ventricles

Sex has a large influence on cardiac electrophysiological properties. Whether sex differences exist in apamin‐sensitive small conductance Ca2+‐activated K+ (SK) current (IKAS) remains unknown. We performed optical mapping, transmembrane potential, patch clamp, western blot and immunostaining in 62 normal rabbit ventricles, including 32 females and 30 males. IKAS blockade by apamin only minimally prolonged action potential (AP) duration (APD) in the basal condition for both sexes, but significantly prolonged APD in the presence of isoproterenol in females. Apamin prolonged APD at the level of 25% repolarization (APD25) more prominently than APD at the level of 80% repolarization (APD80), consequently reversing isoproterenol‐induced AP triangulation in females. In comparison, apamin prolonged APD to a significantly lesser extent in males and failed to restore the AP plateau during isoproterenol infusion. IKAS in males did not respond to the L‐type calcium current agonist BayK8644, but was amplified by the casein kinase 2 (CK2) inhibitor 4,5,6,7‐tetrabromobenzotriazole. In addition, whole‐cell outward IKAS densities in ventricular cardiomyocytes were significantly larger in females than in males. SK channel subtype 2 (SK2) protein expression was higher and the CK2/SK2 ratio was lower in females than in males. IKAS activation in females induced negative intracellular Ca2+–voltage coupling, promoted electromechanically discordant phase 2 repolarization alternans and facilitated ventricular fibrillation (VF). Apamin eliminated the negative Ca2+–voltage coupling, attenuated alternans and reduced VF inducibility, phase singularities and dominant frequencies in females, but not in males. We conclude that β‐adrenergic stimulation activates ventricular IKAS in females to a much greater extent than in males. IKAS activation plays an important role in ventricular arrhythmogenesis in females during sympathetic stimulation

Upregulation of Oxytocin Receptor in the Hyperplastic Prostate

Background: The etiology of benign prostatic hyperplasia (BPH) is complex, both age and androgen are thought to be important. However, the failure of androgen blockade treatments suggests other paracrine/autocrine factors involved in BPH. Oxytocin was found to have a paracrine/autocrine role in prostate in recent years. The influence of BPH on prostatic oxytocin receptor (OTR) expression has never been studied.Material and methods: A testosterone-estradiol induced rat model of BPH was employed and human hyperplastic prostate specimens were harvested. Expressions of OTR, α1-adrenoreceptor subtypes and nitric oxide synthase isoforms were determined via real-time RT-PCR. OTR was further analyzed with Western-Blotting and histological examination. Subsequently, rat epithelial cells, human stromal cells and epithelial cells were cultured in vitro and treated with gradient concentrations of OT from 1 to 5 days. Cell proliferation was tested by Cell Counting Kit-8 and Flow Cytometry.Results: The rat BPH model was validated with significant increased prostate weight. H-E stain revealed a different histopathology between human and rat BPH. Masson's trichrome staining demonstrated that smooth muscle (SM) cells, epithelium cells and collagen fibers were simultaneously augmented in this rat BPH model and human BPH samples. OTR mainly localized in epithelium in rat prostate whereas it mainly localized in stroma in human prostate. OTR gene was upregulated 3.3-fold in rat BPH and 3.0-fold in human BPH, along with increased expression of 2.0-fold α1aARs and 3.0-fold eNOS for rat BPH and 5.0-fold α1aARs for human BPH. The expression of OTR protein was upregulated 1.4-fold in rat BPH and 3.9-fold in human BPH, respectively. Increased concentrations of exogenous OT can accelerate proliferation of rat epithelial cells and human stromal cells but has no impact on human epithelial cells in vitro. Flow Cytometry showed oxytocin could significantly increase G2/M period cell number.Conclusions: Our novel data demonstrates a significant and previously undocumented upregulation of OTR in both rat and human BPH. Moreover, exogenous OT accelerates proliferation of rat prostate epithelial cells and human prostate stromal cells. It is suggested OTR is involved in the development of BPH and OT regulatory system could be a potential new target for the BPH treatment

SAR refinement of antileishmanial N2,N4-disubstituted quinazoline-2,4-diamines

Visceral leishmaniasis is a neglected parasitic disease that has a high fatality rate in the absence of treatment. New drugs that are inexpensive, orally active, and effective could be useful tools in the fight against this disease. We previously showed that N2,N4-disubstituted quinazoline-2,4-diamines displayed low- to sub-micromolar potency against intracellular Leishmania, and lead compound N4-(furan-2-ylmethyl)-N2-isopropyl-7-methylquinazoline-2,4-diamine (4) exhibited modest efficacy in an acute murine model of visceral leishmaniasis. In the present work, thirty-one N2,N4-disubstituted quinazoline-2,4-diamines that had not previously been examined for their antileishmanial activity were evaluated for their potency and selectivity against Leishmania donovani, the causative parasite of visceral leishmaniasis. Quinazoline-2,4-diamines with aromatic substituents at both N2 and N4 exhibited potent in vitro antileishmanial activity but relatively low selectivity, while compounds substituted with small alkyl groups at either N2 or N4 generally showed lower antileishmanial potency but were less toxic to a murine macrophage cell line. Based on their in vitro antileishmanial potency, N4-benzyl-N2-(4-chlorobenzyl)quinazoline-2,4-diamine (15) and N2-benzyl-N4-isopropylquinazoline-2,4-diamine (40) were selected for in vivo evaluation of their pharmacokinetic and antileishmanial properties. While 15 displayed a longer plasma half-life and a greater area under the curve than 40, both compounds showed low efficacy in an acute murine visceral leishmaniasis model. Although the present study did not identify new quinazoline-2,4-diamines with promising in vivo efficacy, the reduced in vitro toxicity of derivatives bearing small alkyl groups at either N2 or N4 may provide clues for the design of safe and effective antileishmanial quinazolines

Model-driven mapping of transcriptional networks reveals the circuitry and dynamics of virulence regulation

Key steps in understanding a biological process include identifying genes that are involved and determining how they are regulated. We developed a novel method for identifying transcription factors (TFs) involved in a specific process and used it to map regulation of the key virulence factor of a deadly fungus—its capsule. The map, built from expression profiles of 41 TF mutants, includes 20 TFs not previously known to regulate virulence attributes. It also reveals a hierarchy comprising executive, midlevel, and “foreman” TFs. When grouped by temporal expression pattern, these TFs explain much of the transcriptional dynamics of capsule induction. Phenotypic analysis of TF deletion mutants revealed complex relationships among virulence factors and virulence in mice. These resources and analyses provide the first integrated, systems-level view of capsule regulation and biosynthesis. Our methods dramatically improve the efficiency with which transcriptional networks can be analyzed, making genomic approaches accessible to laboratories focused on specific physiological processes